NY-ESO-1 peptide derivatives, and uses thereof

ABSTRACT

The invention relates to variant peptides which bind to HLA molecules, leading to lysis of cells via cytolytic T cell lines. The variants are based upon NY-ESO-1 peptides. The peptides can be incorporated into immune tetramers, which are useful as T cell sorters.

FIELD OF THE INVENTION

This invention relates to HLA binding peptides derived from an antigenassociated with cancer. These peptides bind to Class I molecules, andprovoke lysis of the cells to which they bind by cytolytic Tlymphocytes.

BACKGROUND AND PRIOR ART

It is fairly well established that many pathological conditions, such asinfections, cancer, autoimmune disorders, etc., are characterized by theinappropriate expression of certain molecules. These molecules thusserve as “markers” for a particular pathological or abnormal condition.Apart from their use as diagnostic “targets”, i.e., materials to beidentified to diagnose these abnormal conditions, the molecules serve asreagents which can be used to generate diagnostic and/or therapeuticagents. A by no means limiting example of this is the use of cancermarkers to produce antibodies specific to a particular marker. Yetanother non-limiting example is the use of a peptide which complexeswith an MHC molecule, to generate cytolytic T cells against abnormalcells.

Preparation of such materials, of course, presupposes a source of thereagents used to generate these. Purification from cells is onelaborious, far from sure method of doing so. Another preferred method isthe isolation of nucleic acid molecules which encode a particularmarker, followed by the use of the isolated encoding molecule to expressthe desired molecule.

To date, two strategies have been employed for the detection of suchantigens, in e.g., human tumors. These will be referred to as thegenetic approach and the biochemical approach. The genetic approach isexemplified by, e.g., dePlaen et al., Proc. Natl. Sci. USA 85: 2275(1988), incorporated by reference. In this approach, several hundredpools of plasmids of a cDNA library obtained from a tumor aretransfected into recipient cells, such as COS cells, or intoantigen-negative variants of tumor cell lines which are tested for theexpression of the specific antigen. The biochemical approach,exemplified by, e.g., O. Mandelboim, et al., Nature 369: 69 (1994)incorporated by reference, is based on acidic elution of peptides whichhave bound to MHC-class I molecules of tumor cells, followed byreversed-phase high performance liquid chromography (HPLC). Antigenicpeptides are identified after they bind to empty MHC-class I moleculesof mutant cell lines, defective in antigen processing, and inducespecific reactions with cytotoxic T-lymphocytes. These reactions includeinduction of CTL proliferation, TNF release, and lysis of target cells,measurable in an MTT assay, or a ⁵¹Cr release assay.

These two approaches to the molecular definition of antigens have thefollowing disadvantages: first, they are enormously cumbersome,time-consuming and expensive; and second, they depend on theestablishment of cytotoxic T cell lines (CTLs) with predefinedspecificity.

The problems inherent to the two known approaches for the identificationand molecular definition of antigens is best demonstrated by the factthat both methods have, so far, succeeded in defining only very few newantigens in human tumors. See, e.g., van der Bruggen et al., Science254: 1643-1647 (1991); Brichard et al., J. Exp. Med. 178: 489-495(1993); Coulie, et al., J. Exp. Med. 180: 35-42 (1994); Kawakami, etal., Proc. Natl. Acad. Sci. USA 91: 3515-3519 (1994).

Further, the methodologies described rely on the availability ofestablished, permanent cell lines of the cancer type underconsideration. It is very difficult to establish cell lines from certaincancer types, as is shown by, e.g., Oettgen, et al., Immunol. Allerg.Clin. North. Am. 10: 607-637 (1990). It is also known that someepithelial cell type cancers are poorly susceptible to CTLs in vitro,precluding routine analysis. These problems have stimulated the art todevelop additional methodologies for identifying cancer associatedantigens.

One key methodology is described by Sahin, et al., Proc. Natl. Acad.Sci. USA 92: 11810-11913 (1995), incorporated by reference. Also, seeU.S. Pat. No. 5,698,396, and patent application Ser. No. 08/479,328filed Jan. 3, 1996. All three of these references are incorporated byreference. To summarize, the method involves the expression of cDNAlibraries in a prokaryotic host. (The libraries are secured from a tumorsample). The expressed libraries are then immnoscreened with absorbedand diluted sera, in order to detect those antigens which elicit hightiter humoral responses. This methodology is known as the SEREX method(“Serological identification of antigens by Recombinant ExpressionCloning”). The methodology has been employed to confirm expression ofpreviously identified tumor associated antigens, as well as to detectnew ones. See the above referenced patent applications and Sahin, etal., supra, as well as Crew, et al., EMBO J 144: 2333-2340 (1995).

The SEREX methodology has been applied to esophageal cancer samples, andan antigen has now been identified, and its encoding nucleic acidmolecule isolated and cloned. See, e.g., U.S. Pat. No. 5,804,381,referred to supra. The antigen and truncated forms have been found to bereactive with antibodies in the serum of cancer patients. It has alsobeen found that peptides derived form this molecule bind with MHCmolecules, provoking both cytolytic T cell and helper T cell responses.It has been found that variations of these peptides can be used as well.

One difficulty in the area of cancer immunology is a lack of reliableprotocols which can be used to identify and to quantify in vivocytolytic T lymphocyte responses. As a result, it is difficult tocharacterize immune response, and to monitor vaccine trials. It has beenfound that analysis of cytolytic T cells is greatly facilitated by theuse of complexes containing a plurality of T cell targets. Morespecifically, these complexes rely on the known avidity of two bindingpartners, such as avidin or streptavidin and biotin for each other. Itis well known that every molecule of avidin/streptavidin can bind tofour biotin molecules. Constructs where the avidin/streptavidin-biotinsystem is used to form complexes containing multiple targets forcytolytic T cells, i.e., a plurality of immune complexes which comprisean MHC molecule, such as an HLA molecule, a β2 microglobulin, and apeptide which binds to the HLA molecule are taught in, e.g. Ser. No.09/049,850, filed Mar. 27, 1998, and incorporated by reference. Thecomplex is labelled, and can be used to isolate, or to determine,cytolytic T cells of interest in a sample. Such complexes have beenutilized in the invention which follows.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS EXAMPLE 1

Analysis of NY-ESO-1, as discussed in e.g., U.S. Pat. No. 5,804,381,showed that a presenting molecule for this antigen was HLA-A2. Hence, ascreening of the amino acid sequence for NY-ESO-1 was carried out, toidentify all peptides which satisfy this HLA-A2 binding motif, using themodel set forth by D'Amaro et al., Human Immunol. 43: 13-18 (1995), andDrijfhout, et al., Human Immunol. 43: 1-12 (1995) incorporated byreference. Peptides corresponding to all of the amino acid sequencesdeduced thereby were synthesized, using standard techniques, and werethen used in cytotoxicity assays, following Knuth et al., Proc. Natl.Acad. Sci. USA 81: 3511-3515 (1984), incorporated by reference.Specifically, cell line CEMX721.174.T2 (“T2” hereafter), was used,because it expresses HLA-A2, but does not process antigens to MHCcomplexed peptides, thereby making it ideal for experiments of the typedescribed herein. Samples of T2 cells were labelled with 100 uCi ofNa(⁵¹Cr)O₄, using standard methods, and were then washed three times,followed by incubation with 10 ug/ml peptide and 2.5 ug/ml ofβ2-microglobulin. Incubation was for one hour, at room temperature. Thenresponder cells (100 ul of a suspension of CTL NW38-IVS-1) were added,at an effector/target ratio of 90:1, and incubated for four hours in awater saturated atmosphere, with 5% CO₂, at 37° C. Then, plates werecentrifuged at 200×g for five minutes, 100 ul of supernatant wasremoved, and radioactivity was measured. The percentage of ⁵¹Cr releasewas determined in accordance with known strategies. It was found thatthe peptides SLLMWITQCFL (SEQ ID NO: 1), SLLMWITQC (SEQ ID NO: 2), andQLSLLMWIT (SEQ ID NO: 3), were the three best stimulators of HLA-A2restricted NY-ESO-1 specific CTLs. Comparable results were found whenNW-MEL-38 and cell lines SK-MEL-37 and MZ-MEL-19 were used as targets.

EXAMPLE 2

In the next set of experiments, the ability of SEQ ID NOS: 1, 2 and 3 tobind to HLA-A2 molecules and to provoke CTL lysis was confirmed.

Samples of lymph nodes or metastatic lesions were taken from a patientwho presented slowly progressive melanoma with recurrent metastases, allof which were localized to the same paravertebral region. Reversetranscriptase of RNA taken from a subject's tumor indicated that itexpressed NY-ESO-1. Further, the patient's serum indicated high titer ofanti-NY-ESO-1 antibodies.

The surgically resected lymph nodes or metastatic lesions were finelyminced in sterile, RPMI 1640 medium that had been supplemented with 10%fetal calf serum. Suspensions of cells were placed in 24-well tissueculture plates, in 2 ml of Iscove's Dulbecco medium, supplemented with0.24 mM Asn, 0.55 mM Arg, 1.5 mM Gln, 10% pooled human A+ serum, 100U/ml IL-2, and 10 ng/ml of IL-7. The cells were cultured for 2-3 weeks,prior to being assayed via IFN-γ ELISPOT, in accordance with Czerkinsky,et al, J. Immunol. Meth 110:29 (1998), incorporated by reference. Inbrief, 2×10³ cells/well of the short term culture referred to supra werecombined with either 5×10⁴ T2 cells/well, or the same number of T2cells, plus 1 μM of one of SEQ ID NOS: 1-3. Each culture was run induplicate.

A mean number of 19 spots were counted in the control cultures, 424spots with SEQ ID NO: 2,358 spots with SEQ ID NO: 3, and 396 with SEQ IDNO: 1. These elevated numbers correspond to a frequency of about 1NY-ESO-1 specific T cell per 20 tumor infiltrating lymphocytes. Thereactive T cells were then cultured to monoclonality in accordance withValmori, et al, J. Immunol 161: 6956 (1998), incorporated by reference.Five of 24 TIL derived clones derived for the TILs were found to bereactive with NY-ESO-1 derived peptides, when these were tested via CTLassays as described supra.

EXAMPLE 3

In these experiments, CTL ESO5, referred to supra, was tested for itsability to lyse A2⁺ cells which either expressed or did not expressNY-ESO-1. Cell lines NA8-MEL (A2⁺, NY-ESO-1⁻), SK-MEL37 (A2⁺,NY-ESO-1⁺), and Me 275 (A2⁺, NY-ESO-1⁺) were tested, in ⁵¹Cr releaseassays, as described supra.

The results indicated that NA8-Mel was lysed when NY-ESO-1 peptide SEQID NO: 2 was added, but not when it was absent. Presence or absence ofSEQ ID NO: 2 was irrelevant to the lysis of SK-MEL 37 and Me 275, bothof which were lysed under all conditions. These results indicate thatCTL ESO 5 recognizes SEQ ID NO. 2 when exogenously added, and presentedendogenously.

EXAMPLE 4

Experiments were then carried out to determine which of SEQ ID NOS: 1, 2& 3 constituted the optimal T cell epitope for recognition by CTLs. Todetermine this, synthetic peptides corresponding to SEQ ID NOS: 1, 2 and3 were tested in a functional competition binding assay, and then forrecognition by specific CTLs.

The functional competition binding assay employed is that taught byValmori, et al., J. Immunol 161:6956-6962 (1998), incorporated byreference, but elaborated upon herein.

The peptide YMDGTMSQV (SEQ ID NO: 4) is known to bind to HLA-A*0201molecules, and to provoke lysis by an HLA-A*0201 restricted CTL clone,known as LAU 132/2. See Valmori, et al, Canc. Res. 59:2167 (1999). T2cells were labelled with ⁵¹Cr, in the presence of anti class Imonoclonal antibody W6/32. Varying concentrations of SEQ ID NO: 1, 2, or3 (50 μl) were incubated with 50 μl samples of the labelled cells (1000cells/well), for 15 minutes at room temperature. Then, a suboptimal dose(1 nM) of SEQ ID NO: 4 (50 μl) were added, together with 50 μl of the Tcells (5000 cells/well). ⁵¹Cr release was measured after 4 hoursincubation at 37° C. The concentration of each peptide required toachieve 50% inhibition of target cell lysis was then determined as [nM]50%. In order to facilitate the comparison, relative competitiveactivity of each peptide was calculated as [nM] 50% of reference peptideFluMA 58-66 (SEQ ID NO: 5 GILGFVFTL), which is a known high affinityHLA-A*0201 binder, divided by the [nM] 50% value determined for the testpeptides.

The results indicated that SEQ ID NO: 3 was a 100 fold less efficientcompetitor than SEQ ID NO: 4. Further, SEQ ID NO: 2 was 250 fold lessefficient. Surprisingly, given their respective peptide lengths SEQ IDNO: 1 was 10 fold more competitive than SEQ ID NO: 2.

These results suggested that cysteine, as the carboxy terminal aminoacid or SEQ ID NO: 2, was the cause of poor binding to HLA-A2 molecules.To investigate this, three derivatives of SEQ ID NO: 2 were prepared,replacing carboxy terminal cysteine with different hydrophobic aminoacids containing non-polar side chains, such as alanine, leucine, orvaline (SEQ ID NOS: 6-8, respectively). The functional competition assaydescribed supra was carried out with each of these peptides. The resultsare presented in Table 1, which follows. All substitutions clearly anddramatically enhanced peptide binding, for all three substitutedpeptides, indicating that any hydrophobic residue at their positionwould have similar effects. ?

TABLE 1 Relative competitor Peptide Sequence activity Influenza A matrix58-66 GILGFVFTL (SEQ ID NO: 5) 1 NY-ESO-1: 155-163 QLSLLMWIT (SEQ ID NO:3) 0.01 157-167 SLLMWITQCFL (SEQ ID NO: 1) 0.04 157-165 SLLMWITQC (SEQID NO: 2) 0.004 157-C165A SLLMWITQA (SEQ ID NO: 6) 0.4 157-C165LSLLMWITQL (SEQ ID NO: 7) 0.5 157-C165V SLLMWITQV (SEQ ID NO: 8) 10

EXAMPLE 5

As indicated, supra, the peptides described herein were tested forrecognition by specific CTLs. In these experiments, target T2 cells werelabelled with ⁵¹Cr for 1 hour, at 37° C., and then washed two times.Sample (1000 labelled cells in 50 μl) were then incubated with varyingconcentrations of peptide, for 15 minutes. Effector cells (50 μl) werethen added. These effector cells are ESO1 specific CTL clone ESO5. Thelymphocyte: target ratio was 30:1. Chromium release was measured after 4hours of incubation at 37° C., by testing 100 μl supernatant samples.

Specific percent lysis was calculated as:$100 \times \frac{\lbrack ( {{experimental} - {{spontaneous}\quad {release}}} ) \rbrack}{( {{total} - {{spontaneous}\quad {release}}} )}$

The results, which follow in Table 2, are presented as the peptidenanomolar concentration giving 50% maximal activity. Also presented inTable 2 is the “relative antigenic activity” value, which is calculatedas [nM] 50% of the peptide SEQ ID NO: 2 divided by [nM] 50% of the testpeptide. Recognition of the analogue peptides by CTL was comparable to,or better than, the parental peptide. These results demonstrate that,among natural NY-ESO-1 peptides, SEQ ID NO: 2 was the optimallyrecognized antigenic peptide. Further, of the substituted peptides, SEQID NO: 8 was recognized as efficiently as SEQ ID NO: 2, and the otherswere recognized more efficiently. SEQ ID NO: 6 was 1000 fold moreefficiently recognized than SEQ ID NO: 2.

TABLE 2 Peptide Peptide Relative antigenic Sequence ([nM] 50%) activitySLLMWITQC 0.6 1 SLLMWITQA 0.0005 1,200 SLLMWITQL 0.01 60 SLLMWITQV 1 0.6QLSLLMWIT 50 0.012 SLLMWITQCFL 50 0.012

EXAMPLE 6

This experiment describes the basic technique for making tetramericcomplexes of antigen, used in the examples which follow. In order tomake the desired tetramers, it was first necessary to prepare constructswhich would encode modified HLA-A*0201 molecules. To do this, total RNAwas extracted from HLA-A*0201 positive cells, and HLA-A*0201 was thencloned, using specific primers for the molecule, and reversetranscription polymerase chain reaction (RT-PCR). Altman et al., Science274: 94-96 (Oct. 4, 1996) but with a new 3′ primer, i.e.5′-GCAGGATCCCGGCTCCCATCCTCA GGGTGAGGGGC-3′ (SEQ ID NO: 9) incorporatedby reference, was followed. Simultaneously with the RT-PCR, the aminoterminal nucleotide sequence was altered to optimize protein expressionin the vector used. See Garboczi et al., Proc. Natl. Acad. Sci. USA 89:3429 (1992) incorporated by reference. Once this was done, theextracellular coding portion of the molecule was amplified, again usingspecific primers. The resulting construct was recloned into a vectorwhich would produce a BirA biotinylation recognition site in frame atthe 3′-end of the HLA-A*0201 heavy chain. The modified HLA-A*0201 and β2microglobulin were overexpressed in separate E. coli cultures. Theresulting inclusion bodies were purified and the HLA and β2microglobulin recombinant proteins were solubilized into urea, and thenrefolded, in a refolding solution, at 4° C. to form complexes. (Therefolding solution contained 100 mM Tris, at pH 8.0, L-arginine, 400 mM,EDTA, 2 mM, reduced glutathione, 5 mM, oxidized glutathione, 0.5 mM,PMSF, 0.1 mM, HLA heavy chain, and β2 microglobulin 1 μM, and 10 μM ofthe peptide of interest). The refolding solution was concentrated to 7.5ml, using standard techniques. Then, refolding buffer was exchanged withBirA reaction buffer (Tris 100 mM, pH 7.5, NaCl 200 mM, Mg Cl₂ 5 mM,PMSF 100 μM, leupeptin 1 μM, and pepstatin 1 μM), the last three beingadded immediately before use.

The complexes were then biotinylated with biotin holoenzyme synthase(the BirA enzyme) by combining the refold mix containing the HLA-A2complex with 50 μM enzyme, 100 mM biotin in 200 mM Tris, and 100 mMadenosine triphosphate. The mixture was incubated overnight at roomtemperature. The biotinylated complexes were then purified, and combinedwith phycoerythrin-labelled streptavidin, to produce tetramericstructures. These were isolated, and reconstituted in small volumes, ata concentration of 1 mg/ml.

EXAMPLE 7

These experiments were designed to assess the frequency of NY-ESO-1specific T cells. Fluorescent tetramers of biotin, HLA-A2, and peptideswere prepared in accordance with Romero, et al, J. Exp. Med. 188:641(1998), and Altman, et al, Science 274:94 (1996), Example 6, supra, aswell as patent application Ser. No. 09/275,993 filed Mar. 25, 1999 andSer. No. 09/049,850, filed Mar. 27,1998, both of which are incorporatedby reference. As antigenic peptides, Flu Ma 58-66 (SEQ ID NO: 5), andthe ESO-1 derivative using alanine as carboxy terminus (SEQ ID NO: 6)were used. This derivative was selected because of its high affinity,since high affinity binding to HLA-A2 facilitates generation of stabletetramers.

Following tetramer assembly, CTL clones specific for the peptides werecombined with the appropriate tetramers in 20 μl of PBS with 2% FCS, andincubated at room temperature for 1 hour, followed by addition of either20 μl of anti-CD8 antibodies labeled with FITC, or a mixture of anti-CD8antibodies labeled with FITC, and anti-CD45RA antibodies labeled with“CYC.” This mixture was incubated for 30 minutes at 4° C., after whichthe cells were washed in the same buffer as described supra, and thenanalyzed by flow cytometry.

The tetramers of the NY-ESO-1 derivative (SEQ ID NO: 6) specificallystained ESO 5, but did not stain the T cells specific for FluMa, andvice versa.

EXAMPLE 8

These experiments were designed to validate the use of tetramers whichcontain NY-ESO-1 peptides in detecting and isolating NY-ESO-1 specific Tcells.

An enriched CD8⁺ T cell sample which had been stimulated with either SEQID NO: 2, or the alanine terminal substitution (SEQ ID NO: 6).(Stimulation was accomplished by loading autologaus antigens presentingcells with the peptide of interest).

Tetramers were made, as described supra, and used to stain the CD8⁺cells fourteen days following stimulation. Cells were sorted, and testedby IFN-γ ELISPOT, as described supra.

Only CD8⁺ tetramer⁺ cells were found to contain IFN-γ productive cells.

Sorted CD8⁺ tetramer⁺ and CD8⁺ tetramer⁻ cells were expanded for twoweeks via PHA stimulation, and both populations were assayed, in a ⁵¹Crrelease assay, on Me 275 and T2 cells, described supra, which had or hadnot been pulsed with SEQ ID NO: 2.

Only CD8⁺ tetramer⁺ cells were effective in killing both cells which hadbeen pulsed with SEQ ID NO: 2 and cells pulsed with the alanine analog.

EXAMPLE 9

These experiments were designed to determine if NY-ESO-1 antigen isprocessed intracellularly to produce a peptide that is presented byHLA-A2 molecules, which in turn stimulates lysis by CTLs.

Tetramers, as described in example 6, supra, were prepared, using thepeptide of SEQ ID NO: 2. These tetramers were then used to stain asample of cells taken from a patient who had been diagnosed previouslyas having a NY-ESO-1 positive melanoma. See Dunbar, et al., J. Immunol162(12):6959-62(1999) and Ser. No. 09/049,850, filed Mar. 27, 1998,incorporated by reference. In brief, however, peripheral bloodlymphocyte (“PBL”) samples were stained with tetramers for 15 minutes at37° C., followed by washing in PBS/1% FCS at 37° C., followed byincubation with a labelled, anti-CD8 antibody for 30 minutes, on ice.The cells were then washed three times, in ice cold PBS/1% FCS, and werethen analyzed via flow cytometry. Positive cells: were cloned, viapulsing the positive cells with 10 μM of the peptide of SEQ ID NO: 2,and culturing in 1 L-2 (200 u/ml) for 5 days.

Four of these positive CTLs were expanded, and tested in the experimentswhich follow.

EXAMPLE 10

The CTLs described supra were then tested for killing specificity. To doso, samples of a cell line typed previously as being NY-ESO-1 positive,a cell line typed previously as being NY-ESO-1 negative, samples of T2cells which had been pulsed with 1 μM of the peptide of SEQ ID NO: 2,and samples of unpulsed T2 cells were combined with the CTLs, in 1:1 and0.3:1 effector/target ratios. Killing was determined using the ⁵¹Crrelease assay described supra.

The results indicated that both the NY-ESO-1 positive cells, and the T2cells pulsed with SEQ ID NO: 2 were killed, while the others were not,demonstrating that intracellular processing of NY-ESO-1 results in thegeneration of a peptide which is recognized by SEQ ID NO: 2 specificCTLs.

EXAMPLE 11

The experiments in this example were designed to determine whether amixture of different CTLs was responsible for the positive resultsobtained with SEQ ID NOS: 1, 2 and 3, or if a single CTL clonerecognized all peptides. In these experiments, T2 cells were pulsed withone of SEQ ID NO: 2, SEQ ID NO: 1, or a peptide consisting of NY-ESO-1amino acids 157-166, i.e.:

SLLMWITQCF (SEQ ID NO: 11).

SEQ ID NO: 3 was also tested, as was a peptide consisting of the first 8amino acids of SEQ ID NO: 1. Varying concentrations of peptide were usedto pulse T2 cells. As a control, SEQ ID NO: 5 was used. The same ⁵¹Crrelease assay described supra was used. Prior to testing the CTLs, thesewere assayed to determine if they each expressed a single T cellreceptor.

The results indicated that all T cells recognized SEQ ID NOS:2, 3 and11, but failed to recognize SEQ ID NO: 1 or the truncated peptide157-164, i.e., a peptide consisting of the first 8 amino acids of SEQ IDNO: 11. These indicate that all three peptides were recognized by clonalCTLs.

EXAMPLE 12

The experiments set forth in this example were designed to determine theintracellular processing requirements for generating the peptide of SEQID NO: 2.

Cerundolo, et al. Nature 345(6274):449-52(1990) have described mutantprocessing cells which are characterized as having defined blocks in theMHC I processing pathways. These cells were used in these experiments.Specifically, a parental cell line was used, i.e., “line 45,” as well as“line. 174,” which was TAP, LMP2, and LMP7 negative, a transfectant ofline. 174, where the line was transfected with vectors encoding TAP1 andTAP2, and a transfectant of line. 174 which had been transfected withvectors encoding TAP1, TAP2, and LMP7. (The abbreviation “TAP” refers to“transporter associated with antigen processing,” and the abbreviation“LMP” refers to “low molecular mass polyprotein.”)

In addition to the transfectants described supra, all of the cells weretransfected with vaccinia virus constructs which encoded NY-ESO-1, at amultiplicity of infection (M.O.I.) of 5, for 90 minutes.

As controls, equivalent numbers of cells (10⁶ cells) were suspended in50 μl of medium containing 100 μM of lactacysetine, for 1 hour prior toaddition of vaccinia virus constructs. After the 90 minute infectionperiod, cells were washed, and suspended in 5 ml of medium containing 1μM of lactacystin, and grown overnight to allow expression of thevaccinia vector. LMP2 and LMP7 are proteosome subunits.

The cells were then combined with CTLs in a ⁵¹Cr release assay of thetype described supra.

The results indicated that the expression of vaccinia encoded NY-ESO-1in TAP deficient cells failed to sensitize them for CTL lysis. On theother hand, the presence or absence of the proteosome subunits LMP2 andLMP7 did not impair the presentation of an epitope recognized by theCTLs. The controls used support this, because .174/TAP cells did notpresent the peptide of SEQ ID NO: 5, which is known to be dependent onLMP7 for presentation, while transfection with a vector encoding LMP7,or the addition of lactacysteine eliminated the block on presentation.

These results suggest, quite strongly, that a non-proteosomal proteaseis involved in the presentation of the NY-ESO-1 epitope.

EXAMPLE 13

These experiments were designed to determine if peptide analogues of SEQID NO: 2, prepared by modification of the C-terminal cysteine residue,had changed immunogenicity. ⁵¹Cr release assays of the type supra werecarried out, in which the peptide (SEQ ID NO: 2), and target cells werecombined with 200 μM of either dithiothreital (DTT) or TCEP (Tris 2carboxymethyl phosphine).

The results indicate that, in the presence of reducing agent,antigenicity of SEQ ID NO: 2 increased 10 fold. Parallel experimentswere carried out with SEQ ID NO: 8, and there was no increase inantigenicity.

EXAMPLE 14

The results observed in example 13, supra, suggested testing modifiedpeptides. In addition to the peptides of SEQ ID NOS:7 and 8, a peptideof formula:

SLLMWITQI (SEQ ID NO: 12),

was synthesized, as were the following 10 mers:

SLLMWITQCV (SEQ ID NO: 13)

SLLMWITQCF (SEQ ID NO: 11, described supra)

SLLMWITQCI (SEQ ID NO: 14)

SLLMWITQAL (SEQ ID NO: 15)

SLLMWITQAI (SEQ ID NO: 16)

SLLMWITQAF (SEQ ID NO: 17)

Further, SEQ ID NO: 2 was prepared where the C-terminal cysteine wasmodified with an —NH₂—CO—CH₂ sidechain. All of these peptides weretested in CTL assays of the type described supra. The peptides of SEQ IDNOS:7, 8 and 12 all increase recognition by NY-ESO-1 157-165 specificCTLs by about 1000 fold, as compared to SEQ ID NO: 2. The peptide of SEQID NO: 14 was also recognized more efficiently than SEQ ID NO: 2.

EXAMPLE 15

In these experiments, the ability of different peptide analogues tostimulate proliferation of NY-ESO-1 specific CTLs was tested.

In brief, PBLs were taken from two melanoma patients who evidenced hightiters of NY-ESO-1 specific antibodies, using a standard assay. ThesePBLs were then stimulated with either 100 nM or 10 nM, of either SEQ IDNO: 2 or SEQ ID NO: 8 following standard protocols. Over a period of twoweeks, SEQ ID NO: 8 resulted in a 14 fold greater expansion of NY-ESO-1specific CTLs. Over a 3 week period, the expansion was 54 fold greaterthan that obtained using SEQ ID NO: 2. These CTLs were stained withtetramers of the type described supra, using SEQ ID NO: 2, and thestaining was positive, indicating that the CTLs recognized the relevantNY-ESO-1 epitope.

It is noteworthy that 10 nM of SEQ ID NO: 8 stimulated the CTLs, whilelarger amounts of native peptide were in fact necessary. Further, infollow up experiments using standard methodologies, it was ascertainedthat the expanded CTLs described in this example were capable of killingNY-ESO-1 positive tumor cells.

The invention, as will be seen from the preceding disclosure, relates topeptides, of sequence:

SLLMWITQX (SEQ ID NO: 10)

wherein X is any amino acid except cysteine, is preferably a hydrophobicamino acid with a non-polar side chain, such as Ala, Val, Leu, Ile, Pro,Phe, Met, Trp or Gly and is most preferably Ala, Ile, Val or Leu. Thesepeptides may be used therapeutically, via administration to a patientwho is HLA-A2 positive, and expresses NY-ESO-1 in connection with apathology, as well as diagnostically, i.e., to determine if HLA-A2positive cells are present, or if relevant CTLs are present, and soforth.

In an alternate embodiment, X is two amino acids, the first of which iscysteine or alanine, and the second of which is any amino acid.Preferably, the second amino acid is Phe, Ile, Val or Leu. Mostpreferably in this embodiment, the first amino acid is cysteine, and thesecond amino acid is Phe or Ile, as in SEQ ID NO: 11, or 14.

The HLA-A2 molecule is an MHC Class I molecule, and T cells whichrespond to complexes of peptides and class I molecules are generallyCD8⁺ cells.

Exemplary of the peptides defined by the core sequence of SEQ ID NO: IDNO. 10 is the peptide defined by SEQ ID NO: ID NO: 6. This peptide, asindicated, binds to HLA-A2 molecules. Hence it is a “marker” for HLA-A2,as well as a component of peptide/MHC complexes which stimulateproliferation of CTLs, as is described supra. Similarly, peptides whereIle, Leu or Val is the carboxy terminus in the structure of SEQ ID NO:10 can be used.

The peptides may be combined with adjuvants to form therapeuticcompositions. Also a part of the invention are nucleic acid moleculeswhich consist of nucleotide sequences (so-called “mini-genes”) whichencode the peptides of the invention. These mini-genes can beincorporated into expression vectors, in operable linkage with apromoter. Additional constructs which encode more than one peptide ofthe invention, including multiple copies of one or more peptides, arepart of the invention. These constructs may be, e.g. in the form ofrecombinant vectors, or so-called “naked DNA”, i.e. small nucleic acidmolecules encoding the desired peptide or peptides. Similarly,recombinant cells which include the DNA or the vectors, such aseukaryotic or prokaryotic cells, are part of the invention.

The ability of these peptides to bind to HLA molecules makes them usefulas agents for determining presence of HLA-A2 positive cells, such asHLA-A*0201 positive cells, by determining whether or not the peptidesbind to cells in a sample. This “ligand/receptor” type of reaction iswell known in the art, and various methodologies are available fordetermining it.

A further aspect of the invention are so-called “mini genes” which carryinformation necessary to direct synthesis of peptides via cells intowhich the mini genes are, transfected. Mini genes can be designed whichencode one or more antigenic peptides, and are then transferred to hostcell genomes via transfection with plasmids, or via cloning intovaccinia or adenoviruses. See, e.g., Zajac, et al., Int. J. Cancer 71:496 (1997), incorporated by reference These recombinant vectors, such asrecombinant vaccinia virus vectors, can be constructed so as to producefusion proteins. For example, as was shown, supra, fusion proteins canbe constructed where one portion of the fusion protein is the desiredtumor rejection antigen precursor, or tumor rejection antigen, andadditional protein or peptide segments can be included. Exemplary, butby no means the only types of additional protein or peptide segmentswhich can be added to the fusion proteins, are reporter proteins orpeptides, i.e., proteins or peptides which give an observable signal soas to indicate that expression has occurred, such as green fluoresenceprotein. Additional reporter proteins include, but are by no meanslimited to, proteins such as βgalactosidase, luciferase, dhfr, and“eGFP”, or enhanced green fluorescent protein, as described by Cheng, etal., Nature Biotechnology 14:606 (1996), incorporated by reference, andso forth. The various reporter proteins available to the skilled artisancan be, and are used, in different ways. For example, “GFP” and “eGFP”can be used to visualize infected cells, thereby facilitating trackingwhen flow cytometry is used, and the isolation of the cells so infected.Other reporter proteins are useful when methods such as westernblotting, immunoprecipitation, and so forth are used. These techniquesare standard in the art and need not be reiterated here. Protein orpeptide segments which facilitate the cleavage of the tumor rejectionantigen precursor or tumor rejection antigen from the fusion peptide mayalso be included. The fusion protein can include more than one tumorrejection antigen, as described, supra, and can also include proteins orpeptides which facilitate the delivery of the tumor rejection antigen orantigens to a relevant MHC molecule. Such proteins and peptides are wellknown to the art, and need not be elaborated herein.

Also a part of the invention are recombinant cells which have beentransfected with the recombinant reporter vectors described herein. Suchcells may be, e.g., any type of eukaryotic cell, with human cells beingespecially preferred. Such cells can then be used, e.g., to producetumor rejection antigen precursors or tumor rejection antigens. They canalso be used, in an ex vivo context, to generate cytolytic T cellsspecific for particular complexes of MHC molecules and tumor rejectionantigens. This can be done simply by contacting the transfected cells toa source of T cells, such as a blood sample, so as to provoke theproliferation of any cells in the sample specific to the complexes ofMHC molecules and TRAs (i.e., tumor rejection antigens) producedfollowing expression of the fusion protein, and processing of the TRA.Such cells, when rendered non-proliferative, can also be used as vaccinematerials, as they will present the relevant complexes on their surface,and provoke the same type of T cell response in vivo, as is shownherein. Similarly, the vectors can be used as vaccine materials per se,and can be administered to a patient in need of a T cell responseagainst complexes of MHC molecules and peptide on cell surfaces. Ofcourse, T cells generated ex vivo can also be used to treat patients.

The peptides may be combined with peptides that are other tumorrejection antigens to form “polytopes.” Exemplary peptides include thoselisted in U.S. patent application Ser. No. 08/672,351 filed Jun. 25,1996, now abandoned, Ser. No. 08/718,964, now U.S. Pat. No. 5,932,694,Ser. No. 08/487,135 now U.S. Pat. No. 5,821,122, Ser. No. 08/530,569,now U.S. Pat. No. 5,939,526, and Ser. No. 08/880,963, now U.S. Pat. No.6,025,470, all of which are incorporated by reference.

Additional peptides which can be used are those described in thefollowing references, all of which are incorporated by reference: U.S.Pat. Nos. 5,405,940; 5,487,974; 5,519,117; 5,530,096; 5,554,506;5,554,724; 5,558,995; 5,585,461; 5,589,334; 5,648,226; and 5,683,886;PCT International Publication Nos. 92/20356; 94/14459; 96/10577;96/21673; 97/10837; 97/26535; and 97/31017 as well as pending U.S.application Ser. No. 08/713,354, filed Sep. 13, 1996, now U.S. Pat. No.6,265,215.

Polytopes are groups of two or more potentially immunogenic or immunestimulating peptides, which can be joined together in various ways, todetermine if this type of molecule will stimulate and/or provoke animmune response.

These peptides can be joined together directly, or via the use offlanking sequences. See Thompson et al., Proc. Natl. Acad. Sci. USA92(13): 5845-5849 (1995), teaching the direct linkage of relevantepitopic sequences. The use of polytopes as vaccines is well known. See,e.g., Gilbert et al., Nat. Biotechnol. 15(12): 1280-1284 (1997); Thomsonet al., supra; Thomson et al., J. Immunol. 157(2): 822-826 (1996); Tamet al., J. Exp. Med. 171(1): 299-306 (1990), all of which areincorporated by reference. The Tam reference in particular shows thatpolytopes, when used in a mouse model, are useful in generating bothantibody and protective immunity. Further, the reference shows that thepolytopes, when digested, yield peptides which can be and are presentedby MHCs. Tam shows this by showing recognition of individual epitopesprocessed from polytope ‘strings’ via CTLs. This approach can be used,e.g., in determining how many epitopes can be joined in a polytope andstill provoke recognition and also to determine the efficacy ofdifferent combinations of epitopes. Different combinations may be‘tailor-made’ for the patients expressing particular subsets of tumorrejection antigens. These polytopes can be introduced as polypeptidestructures, or via the use of nucleic acid delivery systems. Toelaborate, the art has many different ways available to introduce DNAencoding an individual epitope, or a polytope such as is discussedsupra. See, e.g., Allsopp et al., Eur. J. Immunol. 26(8); 1951-1959(1996), incorporated by reference. Adenovirus, pox-virus, Ty-virus likeparticles, plasmids, bacteria, etc., can be used. One can test thesesystems in mouse models to determine which system seems most appropriatefor a given, parallel situation in humans. They can also be tested inhuman clinical trials.

Also, a feature of the invention is the use of these peptides todetermine the presence of cytolytic T cells in a sample. It was shown,supra, that CTLs in a sample will react with peptide/MHC complexes.Hence, if one knows that CTLs are in a sample, HLA A2 positive cells canbe “lysed” by adding the peptides of the invention to HLA-A2 positivecells, such as HLA-A*0201 positive cells, and then determining, e.g.,radioactive chromium release, TNF production, etc. or any other of themethods by which T cell activity is determined. Similarly, one candetermine whether or not specific tumor infiltrating lymphocytes(“TILs”) are present in a sample, by adding one of the claimed peptideswith HLA-A2 positive cells to a sample, and determining lysis of theHLA-A2 positive cells via, e.g., ⁵¹Cr release, TNF presence and soforth. In addition, CTL may be detected by ELISPOT analysis. See forexample Schmittel et al., (1997). J. Immunol. Methods 210: 167-174 andLalvani et al., (1997). J. Exp. Med. 126: 859 or by FACS analysis offluorogenic tetramer complexes of MHC Class I/peptide (Dunbar et al.,(1998), Current Biology 8: 413-416, Romero, et al., J. Exp. Med. 188:1641-1650 (1998). All are incorporated by reference. To elaborate, thecomplexes comprise a first binding partner and a second binding partner,wherein the first and second binding partner are specific for eachother. These can be, e.g., avidin or streptavidin and biotin, anantibody or a binding portion of an antibody specific to biotin, and soforth. The key feature is that the second binding partner must be boundto a plurality of complexes of an MHC molecule, a β2 microglobulinmolecule and a peptide which binds specifically to said MHC molecule,and the multicomponent complex must be labelled. The MHC molecules arepreferably HLA-A2 molecules; however, it will be understood by theartisan of ordinary skill that any HLA molecule could be used.

Preferably, the second binding partner is biotin, but it may also be,e.g., an antibody which is specific for a component of the HLA/β2microglobulin/peptide complex, such as an HLA specific antibody, or a β2microglobulin specific antibody. Similarly, the first binding partnermay be e.g., recombinant or naturally occurring protein L, recombinantor naturally occurring protein A, or even a second antibody. The complexcan be in soluble form, or bound, e.g., to a removable solid phase, suchas a magnetic bead.

The number of HLA/β2 microglobulin/peptide complexes in the largemolecule of the invention may vary. It comprises at least two complexes,and preferably at least four, but more may be present as well.

The complex of binding partners and HLA/β2 microglobulin/peptide may belabelled, using any of the labels known to the art. Examples offluorescent labels are given supra. Enzymatic labels, such as alkalinephosphatase, metal particles, colored plastics made of syntheticmaterials, radioactive labels, etc., may all be used.

A third binding partner may also be used, which binds, specifically, tothe first binding partner. For example, if the first binding partner isstreptavidin, and the second binding partner is biotin, then the thirdbinding partner may be a streptavidin specific antibody. When three ormore binding partners are used, the label referred to supra may beattached to any of the binding partners so long as engagement with theHLA/β2 microglobulin peptide complexes is not impaired.

The complexes may be used, e.g., to identify or to isolate cytolytic Tcells present in a sample, where these cells are specific for the HLA/β2microglobulin/peptide complex. Such cytolytic T cells bind to theimmunocomplexes of the invention. In a preferred embodiment, the samplebeing tested is treated with a reactant which specifically binds to acytolytic T cell, wherein said label provides a detectable signal. Thesample, including labelled CTLs, is then contacted to the complex, whereit binds, and can be separated via any of the standard, well knownapproaches to cell separation. Preferably, FACS is used, but otherseparation methodologies will be known to the skilled artisan as well.The peptide used is left to the needs of the skilled artisan, and willdepend, e.g., on the nature of the specific MHC system underconsideration.

Additionally, the method can be used to monitor the status of tumors,following administration of a particular therapeutic agent, such as avaccine. Further, since the methodology can be used to identifycytolytic T cell precursors, as shown, supra, one can identifycandidates for potential therapies by determining if they possess therelevant T cell precursors.

Of course, the peptides may also be used to provoke production of CTLs.As was shown, supra, CTL precursors develop into CTLs when confrontedwith appropriate complexes. By causing such a “confrontation” as itwere, one may generate CTLs. This is useful in an in vivo context, aswell as ex vivo, for generating such CTLs.

Also a part of the invention are so-called “cocktails” comprising aplurality of different peptides, at least one of which is a peptide ofthe invention, as well as “polytope” molecules, and nucleic acidmolecules encoding them. “Polytope” as used herein, refers to arecombinant molecule designed to contain a plurality of peptidesequences which are presented by MHC molecules, following intracellularprocessing. Such polytopes can consist of a repeating epitope, a numberof different epitopes, and so forth.

Other features and applications of the invention will be clear to theskilled artisan, and need not be set forth herein.

The terms and expression which have been employed are used as terms ofdescription and not of limitation, and there is no intention in the useof such terms and expression of excluding any equivalents of thefeatures shown and described or portions thereof, it being recognizedthat various modifications are possible within the scope of theinvention.

18 1 11 PRT Homo sapiens 1 Ser Leu Leu Met Trp Ile Thr Gln Cys Phe Leu 15 10 2 9 PRT Homo sapiens 2 Ser Leu Leu Met Trp Ile Thr Gln Cys 1 5 3 9PRT Homo sapiens 3 Gln Leu Ser Leu Leu Met Trp Ile Thr 1 5 4 9 PRT Homosapiens 4 Tyr Met Asp Gly Thr Met Ser Gln Val 1 5 5 9 PRT H. influenzae5 Gly Ile Leu Gly Phe Val Phe Thr Leu 1 5 6 9 PRT Artificial sequenceMutagen 9 Synthesized. Position 9 is mutated from Cys to Ala 6 Ser LeuLeu Met Trp Ile Thr Gln Ala 1 5 7 9 PRT Artificial sequence Mutagen 9Synthesized. Position 9 is mutated from Cys to Leu 7 Ser Leu Leu Met TrpIle Thr Gln Leu 1 5 8 9 PRT Artificial sequence Mutagen 9 Synthesized.Position 9 is mutated from Cys to Leu 8 Ser Leu Leu Met Trp Ile Thr GlnVal 1 5 9 34 DNA Homo sapiens 9 gcaggatccc ggctcccatc ctcagggtga gggc 3410 9 PRT Homo sapiens Mutagen 9 Syntheisized. Xaa is any amino acid,preferably one with a non-polar side chain, such as Ala, Val, Leu, Ile,Pro, Phe, Met, Trp, or Gly 10 Ser Leu Leu Met Trp Ile Thr Gln Xaa 1 5 1110 PRT Homo sapiens 11 Ser Leu Leu Met Trp Ile Thr Gln Cys Phe 1 5 10 129 PRT Artificial sequence Mutagen 9 Synthesized. At position 9, of SEQID NO2,Cys is replaced by Ile 12 Ser Leu Leu Met Trp Ile Thr Gln Ile 1 513 10 PRT Artificial sequence Mutagen 10 Synthesized. At position 10, ofSEQ ID NO 11, Phe is replaced by Val 13 Ser Leu Leu Met Trp Ile Thr GlnCys Val 1 5 10 14 10 PRT Artificial sequence Mutagen 10 Synthesized. Atposition 10 of SEQ ID NO 11, Phe is replaced by Ile 14 Ser Leu Leu MetTrp Ile Thr Gln Cys Ile 1 5 10 15 10 PRT Artificial sequence Mutagen9..10 Sythesized. At positions 9 and 10 of SEQ ID NO 11, Cys Phe isreplaced by Ala Leu 15 Ser Leu Leu Met Trp Ile Thr Gln Ala Leu 1 5 10 1610 PRT Artificial sequence Mutagen 9..10 Synthesized. At positions 9 and10 of SEQ ID NO 11, Cys Phe is replaced by Ala Ile 16 Ser Leu Leu MetTrp Ile Thr Gln Ala Ile 1 5 10 17 10 PRT Artificial sequence Mutagen 9Synthesized. At position 9 of SEQ ID NO 11, Cys is replaced by Ala 17Ser Leu Leu Met Trp Ile Thr Gln Ala Phe 1 5 10 18 10 PRT Artificialsequence Mutagen 9..10 Synthesized.The first Xaa is Cys or Ala. Thesecond Xaa is any amino acid, and is preferablyVal, Phe, Ile or Leu 18Ser Leu Leu Met Trp Ile Thr Gln Xaa Xaa 1 5 10

I claim:
 1. An isolated decapeptide of formula: SLLMWITQXX (SEQ ID NO:18) wherein the first X is cysteine, and the second X is Ile, Val, orLeu.
 2. An isolated decapeptide of formula: SLLMWITQXX (SEQ ID NO: 18)wherein the first X is alanine, and the second X is any amino acid. 3.The isolated decapeptide of claim 2, wherein the second X is Phe, Ile,Val or Leu.
 4. The isolated decapeptide of claim 1, wherein the second Xis Ile (SEQ ID NO: 14).
 5. The isolated decapeptide of claim 1, whereinthe second X is Val (SEQ ID NO: 13).
 6. The isolated decapeptide ofclaim 1, wherein the second X is Leu (SEQ ID NO: 18).
 7. The isolateddecapeptide of claim 3, wherein the second X is Phe (SEQ ID NO: 17). 8.The isolated decapeptide of claim 3, wherein the second X is Ile SEQ IDNO: 16).
 9. The isolated decapeptide of claim 3, wherein the second X isVal (SEQ ID NO: 18).
 10. The isolated decapeptide of claim 3, whereinthe second X is Leu (SEQ ID NO: 15).
 11. A composition useful inprovoking a cytolytic T cell response comprising the isolateddecapeptide of claim 1, and an adjuvant.
 12. A composition useful inprovoking a cytolytic T cell response comprising the isolateddecapeptide of claim 2, and an adjuvant.
 13. The composition of claim 1,further comprising at least one additional tumor rejection antigen. 14.The composition of claim 2, further comprising at least one additionaltumor rejection antigen.